Clarification of protein precipitate suspensions using anionic polymeric flocculants

ABSTRACT

Protein suspensions comprising soluble and insoluble components are purified via flocculation with anionic polymers, such as polyacrylamides, potato starch, or modified cellulose. The procedure improves the efficiency of solid/liquid separations and can minimize or eliminate the requirement for centrifugation and/or filtration in large scale biotechnological processes. The method is particularly well suited for the purification and clarification of protein suspensions containing soluble somatotropin monomer using polyacrylamide and polysaccharide flocculants.

This is a divisional of application Ser. No. 09/354,493, now U.S. Pat.No. 6,307,013, filed Jul. 16, 1999, which is a conversion of provisionalapplication Ser. No. 60/093,555, filed Jul. 21, 1998.

BACKGROUND OF THE INVENTION

The present invention relates generally to the purification of proteinsproduced by recombinant DNA technologies. More particularly, it concernsthe clarification of protein suspensions containing soluble andinsoluble components via flocculation with anionic polymers.

The use of flocculating agents has been described in several industrialsettings, including the biotechnology industry. For example, U.S. Pat.No. 5,047,511 describes the use of cationic flocculating agents in therecovery of recombinant somatotropin protein from a protein solutioncontaining soluble high molecular weight contaminating proteins. Thisinvolved the selective precipitation of contaminating high molecularweight proteins by adding a cationic flocculant containing quaternaryammonium groups and then recovering the somatotropin from the solution.

Somatotropins, also known as growth hormones, are polypeptides producedand secreted by cells of the pituitary gland. These proteins are knownto be effective in promoting pre-adult skeletal growth and meatproduction of beef cattle and swine, and can be produced reliably andinexpensively in large quantities by recombinant DNA technology. Inaddition, they are known to affect a variety of metabolic processesincluding the stimulation of lactation, improvements of the efficiencyof converting feed to meat or milk, lipid-mobilizing effects, andothers.

Recombinant DNA technology provides a means for the large scaleproduction of heterologous proteins of interest in bacterial host cells.In the case of somatotropin, a growth hormone, the protein issequestered in inclusion bodies within the cytoplasm of the host cells.The inclusion bodies can be recovered from the host cell culture bydisrupting the cell so as to release the inclusion bodies, andthereafter collecting the inclusion bodies as a solid pellet bydifferential centrifugation. The inclusion bodies are solubilized in anaqueous solution of a suitable chaotropic agent such as urea orguanidine hydrochloride at an alkaline pH and subsequently naturized bycontact with a mild oxidizing agent to form intramolecular disulfidebonds and to refold the protein into its biologically active, nativeconformation. Methods for the solubilization and naturation ofsomatotropin protein produced by E. coli bacteria are described in U.S.Pat. No. 4,511,502 and U.S. Pat. No. 4,652,630, each of which isincorporated herein by reference.

The somatotropin refold solution obtained from the naturation step (asdescribed for example in U.S. Pat. Nos. 4,511,502 and 4,652,630)comprises an aqueous solution of somatotropin monomers, dimers andhigher oligomers, along with residue and other debris from the hostcells. Of these, the somatotropin monomer is the desired biologicallyactive agent. U.S. Pat. No. 5,182,369, the disclosure of which isincorporated herein by reference, describes the selective precipitationof somatotropin dimer and higher oligomers together with residual hostcell proteins and other contaminating substances from a pH-adjustedsomatotropin refold solution, leaving the desired somatotropin monomeras the primary soluble constituent of the suspension.

Once the somatotropin oligomers and other contaminants have beenselectively precipitated using this approach, it is necessary to removethe precipitated proteins and other insoluble contaminants from thesuspension in order to obtain somatotropin monomers of the desiredpurity. Such liquid/solid separations as those required for thispurification step are employed in most industrial biotechnologicalprocesses and are frequently accomplished via centrifugation and/orfiltration procedures.

Flocculating agents can be employed to improve liquid/solid separationsby aggregating the solids that are present in a protein suspension,thereby increasing the particle size of the solids (for review, seeHalverson and Panzer, 1980). An increase in particle size isparticularly beneficial in centrifugation and sedimentation applicationswhere the particle sedimentation velocity is proportional to the squareof the particle radius. The increased sedimentation velocity thatresults from larger particle sizes can improve productivity in any typeof liquid/solid separation where particle sedimentation velocity is afactor.

SUMMARY OF THE INVENTION

This invention broadly concerns the separation of soluble proteins frominsoluble contaminants via flocculation. More particularly, it relatesto the use of anionic polymeric flocculants in the separation andrecovery of somatotropin proteins.

Therefore, in accordance with one aspect of the present invention, thereis provided a method for separating an aqueous protein suspension ofsoluble somatotropin monomer and insoluble contaminants by adding to thesuspension an anionic polymer in an amount and under conditionseffective to cause the flocculation of the insoluble contaminants. Theflocculated, insoluble, material can be easily separated from thesoluble somatotropin monomer to recover a clarified supernatant ofsoluble somatotropin monomer.

In accordance with another aspect of the invention, there is provided amethod for the recovery of somatotropin monomer which comprises

-   -   obtaining a mixture of somatotropin proteins comprising        somatotropin monomer and somatotropin oligomer in aqueous        solution at a pH greater than about 7;    -   producing a protein suspension containing soluble somatotropin        monomer by precipitating a major portion of the somatotropin        oligomer from the solution while maintaining a major portion of        somatotropin monomer in solution by reducing the pH of the        solution to less than about 6.5;    -   adding to the protein suspension containing soluble somatotropin        monomer an anionic polymer in an amount and under conditions        effective to cause the flocculation of the precipitated        proteins; and    -   separating the flocculated material from the solution of        somatotropin monomer.

In accordance with another aspect of the invention, there is provided anaqueous protein suspension comprising somatotropin monomers,somatotropin oligomers (oligomer as used herein refers to dimers as wellas other multimeric forms of the protein), and an anionic polymer.

Suitable anionic polymers used in accordance with the method of thisinvention include but are not limited to polyacrylamides, particularlythose having charge densities in the range of about 1 to about 20%, andpolysaccharides such as starch.

This invention provides an improved means for separating somatotropinmonomer from the insoluble contaminants that are selectivelyprecipitated from the monomer during recovery of the recombinantprotein. Flocculation of the insoluble contaminants increases theirparticle sizes and therefore their sedimentation velocities, providingimproved productivity in liquid/solid separations.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 illustrates the effect of polymer charge density on theflocculation performance.

FIG. 2 illustrates the effect of pH on the flocculation performance fromabout pH 4.0 to about pH 6.0.

FIG. 3 illustrates the effect of pH on the flocculation performance fromabout pH 4.0 to about pH 4.8.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

This invention provides anionic polymeric flocculants for improving theefficiency of liquid/solid separations by increasing the particle sizeof insoluble contaminants in a protein suspension.

“Protein suspension,” as used herein, represents an aqueous suspension,preferably at an acidic pH (i.e., less than about 7), which comprisessoluble and insoluble components. Typically, but not necessarily, thesoluble component includes the desired protein product (e.g. solublesomatotropin monomer) whereas the insoluble components include theundesired contaminants (e.g. somatotropin oligomers and bacterial celldebris).

“Somatotropin” or “ST” refers to any polypeptide that has biologicalactivity and/or chemical structure similar to that of a somatotropinproduced in the pituitary gland of an animal. Such somatotropins includenatural somatotropins produced by the pituitary somatotropic cells or,alternatively, somatotropins produced by recombinant DNA technology inwhich a somatotropin or a variant derived therefrom is expressed bygenetically transformed prokaryotic or eukaryotic cells, e.g. bacteria(such as E. coli), yeast or algae.

“Refold solution” refers to the stock solution obtained as a result ofthe folding and oxidation in the somatotropin naturation step, asdescribed for example in U.S. Pat. Nos. 4,511,502 and 4,652,630.Subsequent adjustment of the pH of the refold solution to below about6.5, preferably to between about 4.0 and about 6.0, selectivelyprecipitates the insoluble contaminants while leaving the solublesomatotropin monomer substantially unaffected.

“ST protein suspension” refers to the aqueous suspension producedessentially in accordance with U.S. Pat. No. 5,182,369, such that thesuspension has a pH between about 4.0 and about 6.5 and comprisesprimarily soluble somatotropin monomer and insoluble contaminants. Priorto the pH precipitation, the solution generally contains from about 1 toabout 30 grams/liter total protein comprising somatotropin, somatotropinaggregates and E. coli proteins. Approximately 40-60% of the totalprotein is typically precipitated in the pH adjustment step, resultingin an oxidized somatotropin monomer purity greater than about 90% in theliquid fraction.

“Flocculation” refers to the aggregation of insoluble particles causedby the addition of a suitable flocculating agent to a proteinsuspension. By increasing the particle size of the insoluble componentspresent in the suspension, the efficiency of solid/liquid separations,such as by filtration or centrifugation, is improved.

“Clarification” refers to the removal of insoluble contaminants from aprotein suspension, thereby improving the clarity/purity of thesuspension, for example as measured by a decrease in thespectrophotometric absorbance of the suspension at 700 mn.

“Separation” refers to the removal of the aqueous phase, containing thesoluble somatotropin monomers, from the insoluble particles followingflocculation of the ST protein suspension. This removal is accomplishedby any means compatible with the present invention including the commonindustrial methods such as sedimentation and filtration. This separationresults in the recovery of a solution of somatotropin monomers which isessentially free of insoluble contaminants.

“Sedimentation” refers to the settling of the flocculated precipitatedcontaminants, either by centrifugation or by gravity.

The following art, to the extent that it provides exemplary proceduralor other details supplementary to those set forth herein, isspecifically incorporated herein by reference:

-   Garcia, F. A. P., “Protein Precipitation” in: Recovery of Biological    Materials, John Wiley & Sons, 1993.-   Gates et al., “Selecting Agitator Systems to Suspend Solids in    Liquids”, Chemical Engineering, May 24, 1976.-   Halverson, F., Panzer, H. P., “Flocculating Agents” in: Encyclopedia    of Chemical Technology, Volume 10, Third Edition, John Wiley & Sons,    1980, pp 489-523.-   Muhle, K. and Domasch, K. “Stability of Particle Aggregates in    Flocculation with Polymers”, Chemical Engineering Progress 29    (1991).-   Wurzburg O.B., “Modified Starches: Properties and Uses”, CRC Press,    Inc., pp. 4-98.

In one aspect of this invention, there are provided methods for improvedliquid/solid separations of an aqueous ST protein suspension containingsoluble proteinaceous product and insoluble contaminants. This methodinvolves the clarification of a protein suspension by flocculation ofthe precipitated contaminants using anionic polymers. A suitableflocculant is dispersed in a protein suspension under conditionseffective to cause the aggregation of the insoluble proteins and othercontaminants present in the suspension while leaving the soluble productsubstantially unaffected. A number of advantages are provided by theflocculation procedure described herein. First, separation efficiency isimproved due to the fact that the sedimentation velocity of solidsincreases with increasing particle size. This can effectively minimizeor even eliminate the need for centrifugation in a solid/liquidseparation, thereby reducing the capital costs associated with theprocess. Also, the method can improve filtration processes by reducingthe pressure drop that is often observed across filters. This change inhydrodynamic properties can allow for higher flow rates and/or the useof less diatomaceous earth (i.e., filter aid) in filtration processes.

Flocculants for use in this invention include polymers having anioniccharge characteristics effective to cause flocculation of insolublematerial present in a protein suspension. One measure of anionic chargecharacteristic is charge density. Charge density is represented by thepercent of monomer present on a given polymer that possess an anionicchemical group. Charge density can be uniform or non-uniform. For someacrylamide based polymers, the charged group is a carboxyl group that isassociated with integrated acrylate molecules (i.e. acrylic acid in anunprotonated state). Acrylate monomer has nearly the same molecularweight and reactivity as the acrylamide monomer. Therefore,copolymerization with these molecules forms a polymer that has a nearuniform distribution of the carboxyl groups. The molar ratio (expressedas a percentage) of the acrylate monomer to the total moles (of acrylateand acrylamide) is the charge density of that polymer. So a polymerhaving a 10% charge density has an acrylate composition of 10%.

Assessment of the anionic character (i.e. charge density) of anacrylamide polymer made by a copolymerization reaction can be reportedin 2 ways. The first and more common practice is to report the molarpercentage of the acrylate (which contains the anionic charge group) inthe acrylamide-acrylate copolymerization reaction feed. This percentageis referred to as the “theoretical” anioncity or “theoretical” chargedensity. The other method of reporting anionic character is based on atitration method that measures the “total” charge density of the testpolymer. This method includes not only the charge density contributionsfrom the copolymerization reactants, but also includes the anionicitythat results from hydrolysis of the amide group that occurs during thepolymerization reaction. The hydrolysis reaction can convert asignificant number of the amide groups to carboxyl groups, as much as 7%for low charge density polymers (as in the case of the Floerger AN 905PWG polymer). In our experience, it is the total anionicity thatdictates how well a given polymer performs in this process application.“Polymer charge density” or “polymer anionicity” herein is defined asthe total charge density, which includes charge resulting from thecopolymerization reaction (i.e. theoretical charge density), plus thecharge contributions originating from hydrolysis.

The anionic features of the polymeric flocculants can be imparted by anyappropriate chemical constituents present on the polymer. Anionicpolymers containing carboxyl, carboxymethyl, phosphate and sulfatefunctionalities, for example, are particularly well suited for theinvention. Examples of preferred flocculants according to this inventioninclude anionic polyacrylamides and anionic polysaccharides.

Polyacrylamides with carboxyl groups represent one preferred class offlocculants for use in this invention. Those having polymer chargedensities between about 1 and 30%, more preferably between about 1 and20%, and most preferably between about 5 and 12% at neutral pH, havebeen found most suitable.

A flocculant used in this invention can have essentially any molecularweight provided it does not adversely effect the desired flocculation ofthe insoluble contaminants. Higher molecular weights may provideimproved flocculation performance. Typically, the average molecularweight will be in the range from about 100,000 to 50,000,000 or more.Preferably it is greater than about 100,000, more preferably greaterthan about 1,000,000, and most preferably greater than about 10,000,000,provided this does not adversely effect the desired flocculationreaction.

In various embodiment of the invention the anionic polymer is present inthe suspension at a concentration between about 20 and about 30 ppm.

In one preferred embodiment of the invention, the ST protein suspensioncomprises an anionic polymer that has a polymer charge density betweenabout 5% and about 12% and having an average molecular weight greaterthan about 10,000,000.

In another preferred embodiment, the ST protein suspension comprises ananionic polymer that is a polyacrylamide present in an amount from about1 to about 100 ppm. The anionic polymer has a polymer charge density offrom about 5% to about 12% and an average molecular weight greater thanabout 1,000,000.

Another preferred embodiment of the invention comprises, a ST proteinsuspension of having a pH of about 4.5. The ST protein suspension ofthis embodiment comprises an anionic polymer that is a polyacrylamidepresent in an amount of about 25 ppm, having a charge density of about10%, and having an average molecular weight of about 16,000,000.

In a preferred embodiment of this invention, a ST protein suspension isclarified by the above approach. U.S. Pat. No. 5,182,369, the disclosureof which is incorporated herein by reference, describes the selectiveprecipitation of somatotropin oligomers and other contaminants as ameans for the recovery of highly purified somatotropin monomer from arefold solution. This is accomplished by reducing the pH of the refoldsolution from the high level employed for the naturation step (usuallyin excess of pH 10) down to an optimum pH end point value that isgenerally in the range of about 4.0 to about 6.5.

The particles that result from the precipitation step are generallysmall and do not readily settle. Under gravitational conditions, asignificant quantity of the solids stay suspended for an indefiniteperiod of time such that a liquid/solid interface is not visuallyobservable. The characteristically slow sedimentation velocity of thissuspension makes clarification using centrifugation difficult. Depthfiltration has also been used to clarify this process stream, but thereare also disadvantages associated with this type of separation, such asthe expense of additional raw materials and waste handling.

Although additional purification steps may be desired, for example bychromatographic or other methods, the ST monomer recovered in accordancewith this invention is substantially free of residual bacterial proteinand other contaminants and is suitable for administration to the targetanimal by injection or implantation without further purification. Thepurified product preferably contains less than about 10%, morepreferably less than about 5% of somatotropin oligomers. Such oligomersare biologically inactive and do not result in any adverse reaction whenadministered to the target animal.

The mixing conditions used in the flocculation process can effectseveral parameters, as is known in the art. These parameters include thesedimentation rate of the flocs and the clarity of the supernatant. Thepreferred parameters with respect to mixing often represent a compromisedesigned to provide the optimal flocculation conditions possible in theface of frequently conflicting requirements of the individualparameters. For example, and increase in supernatant clarity istypically accompanied by a decrease in the sedimentation rate of theflocs.

Mixing of the polymer solution into the process pool can depend on notonly the size, type, location, orientation and rotational speed of themixer, but also can depend on the rate at which the polymer solution isdelivered. So for a given impeller size, two parameters that can bemanipulated include mixer rotational speed and the rate of polymersolution addition. The skilled individual in the art can readilydetermine the optimal conditions in this regard for a given product andprocess configuration.

A balance must be reached between agitation intensity and shear rate.The design of the mixing system used in accordance with this inventioncan assist in achieving this balance. A mixing impeller design that hasa high pumping capacity and low shear rate is well suited for thisapplication. For example, the Chemineer HE-3 high efficiency impeller(Chemineer—Dayton, Ohio) or the Lightnin A-310 high efficiency mixerimpeller (Lightnin Mixers—Rochester, N.Y.) are suitable choices for thistype of operation.

The mixer should also be capable of keeping the flocs suspended duringthe operation. The minimum level of agitation necessary in aflocculation system is bound by the fluid velocity needed to keep theformed flocs in a “just suspended state”, so that solids are not allowedto settle at the bottom of the tank (Gates et al., 1976).

As alluded to above, two desirable features of the flocculation systemdescribed herein are good mixing and low shear. Tank design can to someextent address both of these concerns. Low shear can be met byminimizing the number of surfaces that cause shear by either creation offluid motion or by surface particle interaction. Proper tank geometryand flocculant feed location will help ensure uniform mixing of theflocculant into the process fluids.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

EXAMPLE 1

The objective of the experiments described herein was to survey andidentify flocculating agents that would aggregate the suspendedparticles from an aqueous suspension containing soluble and insolublecomponents. In this example, aqueous solutions of the flocculating agentwere made up and added to the ST protein suspension. The flocculatingagents were blended into this precipitate suspension using an overheadmixer as described in the Lab-Scale Flocculation Procedure #1. Uponcompletion of the flocculating agent addition, the mixer was stopped andthe flocculated solids allowed to settle. The sedimentation velocity andsupernatant clarity were then measured as described below:

Lab-Scale Flocculation Procedure #1

The experimental lab-scale flocculation equipment was assembled using a4-liter glass beaker, a Lightnin® LabMaster™ mixer with a 6.35 cmdiameter A-310 high efficiency mixer impeller, an Ismatec peristalticpump (model mv-ge) and a container for the polymer solution. The 4-literglass beaker was baffled to enhance vertical mixing in the system. The4-liter glass beaker was designed with glass indentions that ran from 1cm above the bottom of the beaker to just below the lip of the beaker.The pump tubing size was selected based on the required feed flow ratesfor the polymer solution and a ⅛″ stainless steel dip tube was used tofeed the polymer solution. The outlet of this tube was located justbelow and just outside the impeller perimeter so as to ensure efficientmixing of the polymer into the process pool. The Ismatec pump wascalibrated to obtain the desired polymer feed flow rate. A strip of tapewith 1 cm increments marked on it was applied in a vertical position onthe side of the 4-liter beaker to provide a means of measuring the levelof the solid-liquid interface. The impeller was positioned approximately3 to 4 cm above the bottom of the 4-liter beaker. The impeller positionwas fixed throughout a set of experiments by aligning the mixer chuckwith a mark on the impeller shaft.

Two liters of the ST protein suspension were transferred to the 4-literbeaker, and then the mixer was turned on with the rotational speed setat 169 rpm. One liter of the polymer solution was pumped into theprocess pool at a constant rate over a 30 minute time frame. Afterdelivery of the polymer solution into the process pool, the mixer wasallowed to mix the contents for an additional 60 seconds and then themixer was turned off. The settling rate of the flocculated particles andthe supernatant absorbance were then measured. The results for a varietyof flocculating agents are shown in Table 1.

Measurements:

Sedimentation Velocity: Upon completion of the polymer solution additionto the precipitate solution, the mixer was stopped, and the flocculatedsolids were allowed to settle. After the initial momentum of the fluidsdiminished, the flocs began to settle and a visibly discretesolid-liquid interface is formed. Sedimentation velocity was measured bytiming the change in the solid-liquid interface level. The timingstarted immediately after the mixer was stopped and the timer wasstopped at a designated solid-liquid interface level in the glassvessel. The velocity at the interface represents the velocity of theslowest moving particles, which have sufficient concentration to bevisible. The slowest moving particles are rate limiting with respect tocycle time and therefore are the most interesting in terms ofcharacterizing the flocculation process. The accuracy of thismeasurement technique is dependent on the formation of a discerniblesolid-liquid interface and on the sedimentation velocity. The formationof large particles creates a situation where sedimentation rates arefast and where a discernible solid-liquid interface is not formed untilthe solids have settled to the bottom of the container. In such casesthe measurement of sedimentation velocity is difficult and will likelyhave a larger margin of error.

Absorbance @ 700 nm: Measurement of the absorbance at 700 nm (A700) is ameans of quantitatively assess the supernatant clarity achieved from agiven treatment or process. The absorbance of a 1 ml sample of thesupernatant after treatment was measured using a Hewlett Packard UV-VISspectrophotometer (model 8453) and a cuvette with a 1 cm path length.The absorbance value obtained using this method can be converted to atransmittance value as per equation 14:%T=100×exp(−A _(700 nm))Intuitively, transmittance is easy to relate to clarity of thesupernatant since it is directly related to clarity, whereas absorbanceis inversely related to clarity. The clarification process of thisinvention results in a solution having A₇₀₀ less than 0.5, preferablyless than 0.1.

TABLE 1 Survey of flocculating agents Characteristics TheoreticalFlocculant Trade MW_(ave) Anion/ Charged Charge Effectiveness NameManufacturer Composition _((Daltons)) Cationic Group Density (+/−)STA-COTE H-44 A. E. Staley Mfg. Corn Starch − Co. STA-JEL 140 A. E.Staley Mfg. Potato Starch 1.5 M Anionic phosphate + Co. Floerger FA 920Chemtall, Inc. Polyacrylamide 15-20 M Nonionic  0% − Floerger AH 912Chemtall, Inc. Polyacrylamide 15-20 M Anionic hydrolyzed ˜2% +homopolymer Floerger AN 903 Chemtall, Inc. Polyacrylamide 15-20 MAnionic carboxyl  1.5% ++ PWG/GR Floerger AN 905 Chemtall, Inc.Polyacrylamide 15-20 M Anionic carboxyl  3% ++ PWG/GR/VHM Floerger AN910 Chemtall, Inc. Polyacrylamide 15-20 M Anionic carboxyl 10% ++ PWGFloerger AN 923 Chemtall, Inc. Polyacrylamide 15-20 M Anionic carboxyl20% + PWG Floerger AN 934 Chemtall, Inc. Polyacrylamide 15-20 M Anioniccarboxyl 30% + PWG Agefloc WT601 Ciba Polyacrylamide 15-20 M Anioniccarboxyl 10% + Agefloc WT611 Ciba Polyacrylamide 15-20 M Anioniccarboxyl 10% ++ CYSEP 2411 CYTEC, Inc. ˜6 M Cationic − CYSEP 2709 CYTEC,Inc. ˜4 M Cationic − CYSEP 329 CYTEC, Inc. Polyamine ˜50 K Cationic −CYSEP 349 CYTEC, Inc. Polyamine ˜250 K Cationic − CYSEP 4022 CYTEC, Inc.PolyDADM ˜50 K Cationic − CYSEP 615 CYTEC, Inc. Polyamine ˜30 K Cationic− Methocel A4M Dow Chemical Co. Methcellulose Nonionic − PREM MethocelE4M Dow Chemical Co. Hydroxypropyl Nonionic − PREM MethocelluloseMethocel F4M Dow Chemical Co. Hydroxypropyl Nonionic − PREMMethocellulose Methocel K100M Dow Chemical Co. Hydroxypropyl Nonionic −PREM Methocellulose Chitosan SIGMA Chitosan ˜370 K Preastol 2500 TRStockhausen, Inc. Polyacrylamide 15-20 M Nonionic  0% − Preastol 2505Stockhausen, Inc. Polyacrylamide 15-20 M Anionic Carboxyl  5% + Preastol2515 TR Stockhausen, Inc. Polyacrylamide 15-20 M Anionic Carboxyl 15% ++Preastol 2520 Stockhausen, Inc. Polyactylamide 15-20 M Anionic Carboxyl20% + “+” = clear supernatant (% Transmittance ≧ 70%), slowsedimentation (velocity < 500 cm/hr) “++” = clear supematant (%Transmittance ≧ 95%), fast sedimentation (velocity ≧ 500 cm/hr); “NT” =not tested; “−” = no visible formation of a solid-liquid interface; “M”= millions; “K” = thousands; MW = molecular weight

These results show that only anionic flocculating agents produceddetectable signs of flocculation as indicated by the sedimentationvelocity of the flocculated solids and by the clarity (reported as %transmittance) of the aqueous phase (supernatant). Flocculating agentsproducing flocs that exhibited sedimentation velocities greater than 500cm/hr and supernatants with % transmittance greater than 95% were givena rating of “++”. Flocculating agents producing flocs that exhibitedsedimentation velocities less than 500 cm/hr and supernatants with %transmittance greater than 70% were given a rating of “+”. Flocculatingagents producing no visible signs of flocculation, as indicated by thelack of formation of a liquid-solid were given a rating of “−”. Itshould be noted that the reported charge densities for the respectiveflocculating agents are theoretical charge densities, and do notrepresent the total charge density.

EXAMPLE 2

A range of flocculating agent concentrations was evaluated usingLab-Scale Flocculation Procedure #2. A high molecular weight (roughly 16million daltons) acrylamide polymer with anionic character was used inthis evaluation. The three primary measurements of interest in theseexperiments were supernatant absorbance, total protein concentration andbST concentration. Table 2 displays the data obtained using thisprocedure. The final polymer concentration referred to herein representthe total polymer concentration after all of the polymer solution hadbeen added to the ST protein suspension.

Lab-Scale Flocculation Procedure #2

-   -   1. Make up the flocculant solution at room temperature to the        desired concentration (i.e. 200 ppm) 1 hour in advance of the        flocculation experiments.    -   2. Label 3 polycarbonate conical-bottom test tubes (w/screw        caps). Each polymer concentration was evaluated in triplicate.    -   3. Pipette the ST protein suspension into each test tube.        Pipette the corresponding volume of the polymer solution into        the ST protein suspension and then immediately cap the test tube        and invert 20 times.    -   4. Place the test tubes into a bucket type centrifuge and spin        at room temperature for 5 minutes at 1000 rpm (Centrifuge type:        Sorvall RC5C, Rotor size: SS34, RCF˜119).    -   5. After centrifugation, gently remove the cap and using a        transfer pipette pull the top 6 ml (of 10 ml total) of        supernatant. Draw liquid near the liquid/air interface while        trying to avoid floating solids. Transfer the supernatant to a        labeled test tube.    -   6. Measure the absorbance of the supernatant at 700 nm. Then        dilute 1 ml of the supernatant in 5 ml of 1% acetic acid.        Measure the absorbance at 280 nm and use the supernatant for        HPLC analysis to determine bST concentration.

Repeat steps 2 through 6 for a range of polymer concentrations.

Measurements:

Total Protein Concentration: The total protein concentration wascalculated from a measured absorbance at a wavelength of 278 nm on adiluted supernatant sample. Solutions of bST have an extinctioncoefficient (extinction coefficient, ε, or molar absorptivity is definedas the constant of proportionality in Beer's law: A=εbc, where A isabsorbance, b is pathlength, and c is the molarity of the absorbingspecies) of 0.651. The supernatant sample was diluted in 1% acetic acidto obtain a total protein concentration near 1.5 grams/liter. The use ofacetic acid as the dilution buffer serves to dissolve any suspendedsolids not removed in the treatment. (The suspended solids not removedin the treatment are considered part of the supernatant. These solidsmust be dissolved so they do not interfere with the absorbancemeasurement at 278 nm.) For this measurement, a 1% acetic acid solutionwas used as the zero reference.

bST Concentration: A portion of the diluted sample from the totalprotein concentration measurement was placed into a vial and analyzedusing reverse phase HPLC.

The first row of data in Table 2 are the data associated with a sampleof the ST protein solution that was filtered through a 0.2 micronsyringe filter. The syringe filter samples sample serves as a means toassess the interaction, if any, of the flocculating agent with thesoluble ST protein. The second row of data in Table 2 are dataassociated with the “control”. The control is a sample of ST proteinsuspension to which flocculating agent was not added.

TABLE 2 Effect of Flocculant Polymer Dosage on the CentrifugalClarification of pH Adjusted ST Protein Suspension Supernatant PolymerSupernatant Protein bST Concentration Absorbance ConcentrationConcentration Yield (ppm) (A700) (g/L)* (g/L)* (%) 0 0.01 8.50 7.72 100%0 1.67 13.48 8.71 113% 1 1.65 12.53 8.62 112% 10 1.32 8.97 8.12 105% 500.15 8.13 8.04 104% 100 0.06 7.87 8.15 106% 500 0.30 6.93 6.75 87% 10000.67 2.35 3.18 41% *Note: Protein and bST concentrations are correctedfor dilutions (i.e. they reflect predilution values).The data show that the total protein concentration decreased, as did thesupernatant absorbance, upon addition of the flocculating agent forfinal flocculating agent concentrations greater than 1 ppm, whencompared to the control, while the soluble ST concentration remainedrelatively constant for samples that were treated with finalflocculating agent concentrations less than 500 ppm. From these data wecan conclude the following: 1) The flocculating agent did not exhibitsignificant interaction with the soluble ST protein so as to remove thesoluble protein from solution for final flocculating agentconcentrations less than 500 ppm. 2) The decrease in supernatantabsorbance and the decrease in the total protein concentration indicatesthat the addition of the flocculating agent to the solution containingsoluble ST protein and precipitated contaminants improved thesedimentation properties of the solids in this system. The increase insedimentation performance is directly related to the size of theparticles in this system, which can be attributed the flocculationactivity of the flocculating agent. Flocculation as measured bymonitoring the total protein concentration was observed to occur forflocculating agent concentrations as low as 1 ppm. Flocculating agentconcentrations above 50 ppm yielded purities at or above 95%, but thebST concentration began to decrease for flocculating agentconcentrations greater than about 100 ppm by this procedure. Thus,although a preferred range of polymer concentrations based on theseexperiments was between about 5 and 100 ppm, the invention is operableat up to 1000 ppm or more of polymer. The observed decrease in bSTconcentration, at polymer concentrations greater than or equal to 500ppm, may be an indication that the flocculant agent was interacting withbST and pulling it down with the other solids.

EXAMPLE 3

Polyacrylamide flocculating agents manufactured by Chemtall Inc.(Riceboro, Ga.) were evaluated. The trade name for the Chemtallflocculating agent tested in this example is Floerger AN 905 GR. Sixlab-scale flocculation experiments were run each using 2 liters of STprotein suspension and 1 liter of flocculant solution. The flocculantsolution was added to a mixed ST protein suspension at a constant flowrate over an approximate time frame of 30 to 40 minutes. The mixingconditions were held constant for this set of experiments so that theeffect of the polymer concentration on the flocculation performancecould be assessed. The sedimentation velocity was approximated bymeasuring the time it took for the liquid/solid interface to reach afixed level in the glass beaker after the over-head mixer was turnedoff. Supernatant absorbance was measured using a spectrophotometer.Table 3 illustrates flocculation performance data for a range ofFloerger An 905 PWG flocculant concentrations.

Table 3 shows the results generated when polymeric acrylamide flocculantwith an average molecular weight of about 16 million daltons and acharge density of 10% (Floerger AN 905 GR [Lot # 8S36839]) was added tothe ST protein suspension to obtain final polymer concentrations rangingfrom 5 to 35 ppm. The mixer speed was 169 rpm, the tank diameter was15.9 cm, temperature was about 20° C., mixer impeller pump capacity was24 liters/minute, impeller diameter was 6.35 cm, and impeller tip speedwas 56 cm/sec, with center mixing in a baffled container.

The data show that the sedimentation velocity increased with an increasein the final polymer concentration, while the supernatant transmittancedecreased with an increase in final polymer concentration. The increasein sedimentation velocity is an expected outcome in that more polymer isavailable to form larger aggregates. The larger aggregates will settlefaster as a result of their larger size.

TABLE 3 Effect Polymer Concentration on the Flocculation PerformanceFlocculation Performance Polymer Initial bST Volume PolymerSedimentation Supernatant Supernatant Volume [AN905]₀ Added [AN905]_(f)Addition Velocity Absorbance Transmittance Exp. # (L) (ppm) (L) (ppm)Time (min) (cm/hr) (λ= 700 nm) (%) 1 2 15 1 5 30 77 0.011 99% 2 30 780.016 98% 3 30 85 0.003 100%  Average = 80 99% Std. Dev. = 4 0.63%   4 245 1 15 30 989 0.026 97% 5 30 1147 0.022 98% 6 30 1042 0.022 98% Average= 1059 98% Std. Dev. = 80 0.25%   7 2 75 1 25 30 1205 0.040 96% 8 311266 0.039 96% 9 30 1456 0.031 97% Average = 1309 96% Std. Dev. = 1310.45%   10 2 105 1 35 30 1603 0.047 95% 11 30 1767 0.052 95% 12 30 22310.038 96% Average = 1685 96% Std. Dev. = 116 0.70%   Sedimentationvelocity data point was not included in the average or std. dev. becausethe solids did not suspend at during the post mixing stage of the run.The flocculated contents were mixed for 1 minute after completion of thepolymer addition.

EXAMPLE 4

Data on the effect of the polymer charge density on the flocculationperformance is listed in Table 4. Two final polymer concentrations (15ppm and 30 ppm) were evaluated using Chemtall flocculant polymers. Thepolymers used in this set of experiments had theoretical chargedensities ranging from 0% to 20%. The reaction conditions were asfollows: ST protein suspension volume=2 liters, mixer size, capacity,and speed as in EXAMPLE 3, tank diameter=15.9 cm, ⅛″ dip tube, volume ofpolymer added=1 liter resulting in a 50% increase in volume.

TABLE 4 Summary of Chemtall Flocculant Evaluations FlocculationPerformance Actual Initial Theoretical Measured polymer Pumpsedimentation Supernatant Exp. Anionicity Anionicity [F]_(o) [F]_(f)addition speed velocity absorbance Transmittance # Flocculant (%) (%)(ppm) (ppm) time (min) setting (cm/hr) (λ = 700 mn) (%) 1 Floerger FA920 0 1 45 15 31 500 0 1.297 27% 2 Floerger AN 903 1.5 6 45 15 32 500785 0.053 95% PWG 3 Floerger AN 905 3 10 45 15 32 510 947 0.034 97% PWG4 Floerger AN 910 10 14 45 15 32 520 275 0.017 98% PWG 5 Floerger 923 AN20 27 45 15 31 530 23 0 026 97% PWG 6 Floerger FA 920 0 1 90 30 0 1.31327% 7 Floerger AN 903 1.5 6 90 30 31 540 1284 0.050 95% PWG 8 FloergerAN 905 3 10 90 30 31 540 1657 0.024 98% PWG 9 Floerger AN 910 10 14 9030 32 540 391 0.002 100% PWG 10 Floerger 923 AN 20 27 90 30 550 25 0.02697% PWG

The data in Table 4 was generated using Chemtall flocculating agents.These data were obtained using the Lab-Scale Flocculation Procedure #1.The sedimentation velocity and the supernatant transmittance data wereplotted against the total polymer anionicity (as measured by themanufacturer), see FIG. 1. Table 4 lists data of the flocculationperformance parameters with the actual measured polymer anionicity. Onlyanionic polymers manufactured using the acrylate and acrylamidepolymerization were used to produce the data in Table 4 (as opposed tousing anionic polymers that were made by hydrolysis). The theoreticalanionicity is calculated by the amount of acrylate used in thecopolymerization reaction. The total anionicity includes the theoreticalanionicity, plus the anionicity resulting from hydrolysis.

The data in Table 4 show that polymeric flocculants that have ananionicity of 1% or lower did not exhibit detectable signs offlocculation as evidenced by the lack of formation of a visiblesolid-liquid interface at static conditions. The data pointscorresponding to a 1% anionicity represent the performance of a polymerthat was manufactured as a nonionic polymer, that is, a polymer withoutcarboxyl groups. The 1% anionic character of this polymer is the resultof acid hydrolysis that occurred during the manufacturing process. Theanionic character of a polymer containing carboxyl groups issignificantly reduced at a low pH. This fact implies that a 1% anioniccharge on the polymer at neutral pH (pH˜7.0) is essentially neutralized(i.e. charge density≈0) at pH 4.5. For purposes of this discussion, thepolymer associated with the 1% anionicity data points is considered ashaving a nonionic character. Formation of a solid-liquid interface didnot occur at static conditions which suggests this polymer did notsignificantly affect the size of the precipitate particles in thesuspension. Measurement of the solution clarity was essentiallymeasurement of the diluted suspension since a separation of solids fromthe liquid did not occur, which explains the low transmittance valueindicated in Table 4.

Flocculations performed using polymers with charge densities greaterthan 1% exhibited a discernible solid-liquid interface. The data clearlyshow that there exists an optimum anionicity for flocculation of thesolids in this system. The 10% anionic polymer exhibited the fastestsedimentation velocity, which indicates that the optimum anionicity isnear 10%. Sedimentation velocities obtained using polymers with chargedensities less than or greater than the 10% anionic polymer weremeasurably slower than the velocity obtained when using the 10% anionicpolymer, while the supernatant clarity appears to be relativelyconsistent for all flocculations performed using polymers with anioniccharacter.

A polymeric flocculant with a charge density less than the optimalcharge density (i.e. the 6% anionic polymer) exhibited a slowersedimentation velocity than the velocity achieved when using the 10%anionic polymer. The data indicate that charge densities less theoptimal charge density result in less efficient flocculation of thesolids.

EXAMPLE 5

The effect of the pH of the pH-adjusted ST protein suspension onflocculation performance was evaluated. The total protein concentrationof refold concentrate was adjusted to approximately 20 g/L usingpurified water. The pH of the diluted refold solution was adjusted using5% acetic acid to 4 different pH endpoints. At each pH a 2 liter sampleof the resulting suspension was taken and further mixed for at a minimumof 1 hour using a stir bar and stir plate. Each of the suspensionsamples were flocculated using the Lab-Scale Flocculation Procedure #1with a 1 liter aliquot of a 75 ppm solution of Floerger AN 905 PWGflocculating agent (Chemtall Inc., 10% anionicity). The dip tubediameter was ⅛″, the mixer rotational speed was 169 rpm. The pHadjustment and the flocculations were run at room temperature (˜23° C.).The pH data and the flocculation performance data is listed in theTables 5 and 6 below. These data indicate that for the Floerger AN 905PWG polyacrylamide flocculant (F905), the preferred pH of thepH-adjusted ST protein suspension is between about 4 and 5, morepreferably between about 4.2 and 4.6.

TABLE 5 Flocculation Dependence on pH Flocculation Performance InitialPolymer Sedimentation Supernatant Pump speed addition time velocityAbsorbance Exp. # pH setting Q_(P) (ml/min) (min) (cm/hr) (700 nm) % T 14.73 600 37 27 504 0.032 97 2 4.60 600 37 27 932 0.053 95 3 4.50 600 3727 1219 0.026 97 4 4.40 600 37 27 1477 0.023 98 5 4.30 600 37 27 15670.025 98

TABLE 6 Flocculation Dependence on pH Flocculation Performance InitialPolymer Sedimentation Supernatant Pump speed addition time velocityAbsorbance Exp. # pH setting Q_(P) (ml/min) (min) (cm/hr) (700 nm) % T 14.80 810 31 32 104 0.014 99 2 4.60 810 31 32 686 0.016 98 3 4.50 810 3132 960 0.013 99 4 4.40 810 31 32 1527 0.016 98 5 4.30 810 31 32 12920.009 99

The data in Tables 5 and 6 show that flocculation performance, asmeasured by sedimentation velocity, is strongly dependent on solutionpH. These data are represented in graphical form in FIGS. 2 and 3. Thesedimentation velocity significantly increases as the pH decreases from4.8 to 4.3. The clarity, as measured by % transmittance, of thesupernatant in this pH range is relatively constant. The data in Tables5 and 6 also indicate that for high molecular weight polymer of about16,000,000 daltons, with a measured charge density of about 10%, theflocculation performance as measured by the sedimentation velocity isoptimal near about pH 4.40.

EXAMPLE 6

A modified version of Lab-Scale Flocculation Procedure #2 was used toevaluate starch and cellulose polymers as flocculating agents.Essentially the same procedure was used as above except for the processvolumes, beaker sizes and polymer addition times. In this experiment 1liter of polymer was added to 1 liter of ST protein suspension over a 1to 2 hour time frame.

Starch polymers were provided by A. E. Staley Manufacturing Company(Decatur, Ill.). One potato starch (Sta Jel 140, prejelatenized potatostarch) were evaluated. The potato starch exhibited visual signs ofparticle aggregation under the conditions tested.

Sta Jel 140 is a prejelatenized potato starch, a starch which isprimarily composed of amylose and amylopectin polymers. Amylose islinear polymer, whereas the amylopectin is a highly branched polymer.Most starches contain 18 to 28% amylose, with potato starch exhibitingamylose content at the lower end of this range (Wurzburg). Potato starchcontains approximately 20% amylose and 80% amylopectin, and has anapproximate average molecular weight of 1.5 million daltons. This ratioof amylose to amylopectin suggests that potato starch is a highlybranched polymer. Unmodified potato starch naturally contains 0.08%phosphorous, which gives this starch its anionic characteristics.

Potato feed starch concentrations of 0.02% (wt/wt) and 0.2% (wt/wt) weretested to obtain final starch polymer concentrations of 0.01% (wt/wt)and 0.1% (wt/wt), respectively. In both cases the Sta Jel 140 potatostarch exhibited visual signs of flocculation. The solids settled atgravitational conditions leaving a nearly crystal clear supernatant. Theformed flocs were smaller than the flocs formed using the Floerger AN905 PWG polymer, which resulted in slower sedimentation of the solids.Measurement of the total soluble protein indicated that there was nodetectable loss in soluble protein concentration upon treatment of theST protein suspension with the potato starch. This is evidence that thepotato starch did not interact with the soluble bST protein.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied in the steps orin the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

1. An aqueous somatotropin (ST) protein suspension comprisingsomatotropin monomers, somatotropin oligomers, and an anionic polymerhaving a polymer charge density of between about 1% and 30%; wherein theST monomers are soluble, wherein the ST oligomers are precipitated, andwherein the anionic polymer and the oligomers are aggregated.
 2. The STprotein suspension of claim 1, wherein the anionic polymer is apolyacrylamide.
 3. The ST protein suspension of claim 2, wherein thepolyacrylamide has a polymer charge density between about 5% and about12%.
 4. The ST protein suspension of claim 2, wherein the polyacrylamidehas a polymer charge density between about 8% and about 11%.
 5. Anaqueous somatotropin (ST) protein suspension comprising ST monomers, SToligomers, and an anionic polymer; wherein the ST monomers are soluble,wherein the ST oligomers are precipitated wherein the anionic polymerand the precipitated oligomers are aggregated, and wherein the anionicpolymer is a polysaccharide.
 6. The ST protein suspension of claim 5,wherein the anionic polymer is starch or modified cellulose.
 7. The STprotein suspension of claim 5, wherein the polysaccharide is potatostarch.
 8. The ST protein suspension of claim 1, wherein the anionicpolymer is present in the suspension at a concentration between about 1and about 1000 ppm.
 9. The ST protein suspension of claim 1, wherein theanionic polymer is present in the suspension at a concentration betweenabout 10 and about 100 ppm.
 10. The ST protein suspension of claim 1,wherein the anionic polymer is present in the suspension at aconcentration between about 20 and about 30 ppm.
 11. The ST proteinsuspension of claim 1, wherein the anionic polymer's average molecularweight is greater than about 100,000.
 12. The ST protein suspension ofclaim 1, wherein the anionic polymer's average molecular weight isgreater than about 1,000,000.
 13. The ST protein suspension of claim 1,wherein the anionic polymer's average molecular weight is greater thanabout 10,000,000.
 14. The ST protein suspension of claim 1, wherein theanionic polymer has a polymer charge density between about 5% and about12% and an average molecular weight greater than about 10,000,000. 15.The ST protein suspension of claim 1, wherein the somatotropin is bovinesomatotropin.
 16. The ST protein suspension of claim 1, wherein theanionic polymer is a polyacrylamide present in an amount from about 1 toabout 100 ppm having a polymer charge density from about 5% to about 12%and an average molecular weight greater than about 1,000,000.
 17. The STprotein suspension of claim 1, wherein the pH of the protein suspensionis about 4.5, and the anionic polymer is a polyacrylamide present in anamount of about 25 ppm, having a charge density of about 10%, and anaverage molecular weight of about 16,000,000.
 18. The ST proteinsuspension of claim 5, wherein the anionic polymer is present in thesuspension at a concentration between about 10 and about 100 ppm. 19.The ST protein suspension of claim 5, wherein the anionic polymer'saverage molecular weight is greater than about 100,000.
 20. The STprotein suspension of claim 5, wherein the anionic polymer's averagemolecular weight is greater than about 1,000,000.